A peer-reviewed open-access journal PhytoKeys 75: 31–55 (2016) Borneocola (Zingiberaceae), a new genus from Borneo 31 doi: 10.3897/phytokeys.75.9837 RESEARCH ARTICLE http://phytokeys.pensoft.net Launched to accelerate biodiversity research Borneocola (Zingiberaceae), a new genus from Borneo Yen Yen Sam1, Atsuko Takano2, Halijah Ibrahim3, Eliška Záveská4, Fazimah Aziz5 1 Forest Research Institute Malaysia, 52109 Kepong, Selangor, Malaysia 2 Museum of Nature and Human Activities, Hyogo 6 chome, Yayoigaoka, Sanda, Hyogo 669-1546, Japan 3 Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia 4 Institute of Botany, University of Innsbruck, Austria 5 Department of Aquatic Science, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia Corresponding author: Yen Yen Sam ([email protected]) Academic editor: Pavel Stoev | Received 12 July 2016 | Accepted 9 November 2016 | Published 29 November 2016 Citation: Sam YY, Takano A, Ibrahim H, Záveská E, Aziz F (2016) Borneocola (Zingiberaceae), a new genus from Borneo. PhytoKeys 75: 31–55. doi: 10.3897/phytokeys.75.9837 Abstract A new genus from Borneo, Borneocola Y.Y.Sam, is described here. The genus currently contains eight spe- cies previously classified as members of the Scaphochlamys Baker. The finding is based on the results of the morphological and molecular studies of Scaphochlamys throughout its geographical range and its closely allied sister groups, Distichochlamys M.F.Newman and Myxochlamys A.Takano & Nagam. Borneocola is nested within the tribe Zingibereae and its monophyly is strongly supported by both ITS and matK se- quence data. The genus is characterised by several thin, translucent and marcescent floral bracts, absence of coloured streaks on the labellum and capitate stigma with two dorsal knobs. The genus is distributed in northwest Borneo and all species are very rare and highly endemic. Keywords Distichochlamys, Myxochlamys, Scaphochlamys, morphology, phylogeny, taxonomy Copyright Yen Yen Sam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 32 Yen Yen Sam et al. / PhytoKeys 75: 31–55 (2016) Introduction Southeast Asia is the centre of diversity for the family Zingiberaceae. Here, new taxa are continuously being discovered and named, both at the generic and specific levels. Several of the recent discoveries were further supported by the phyloge- netic analyses which give a better understanding of the evolutionary relationships within the family (Kress and Larsen 2001; Kress et al. 2010; Leong-Škorničková et al. 2011). During the revision of the genus Scaphochlamys throughout its entire geographical range by the first author, some distinctive morphological traits were observed in several Bornean species, suggesting they might represent a separate group from the Peninsular Malaysian taxa. This hypothesis was confirmed by the phylogenetic analyses which are presented here and the eight species previously included in the genus Scaphochlamys are recircumscribed in this paper as a new genus, Borneocola Y.Y.Sam. The genus Scaphochlamys was described by Baker (1892) in the Flora of British India with Scaphochlamys malaccana Baker from Mt. Ophir (now known as Gunung Ledang), Peninsular Malaysia, chosen as the type species. Holttum (1950) carried out the first comprehensive revision of the genus in which he recognised 19 species, all of which were recorded in the peninsula. When Smith (1987) reviewed the tribe Hedychieae in Borneo, she applied the generic delimitation defined by Holttum and recognised fiveScaphochlamys species in Borneo. Out of the five,S . polyphylla and S. petiolata were formerly placed in the genus Haplochorema K.Schum. Sakai and Nag- amasu (2006) discovered that H. gracilipes K.Schum. also have the characteristics of Scaphochlamys and effected the transfer. Recent years have seen a surge in the new species discovered from Borneo bringing the total number of Bornean Scaphochla- mys to 14 (Poulsen and Searle 2005, Meekiong et al. 2011, Ooi and Wong 2014; Meekiong 2015). Distichochlamys M.F.Newman and Myxochlamys A.Takano & Nagam. are sister genera to Scaphochlamys with several unique characteristics clearly separating them from Scaphochlamys (Newman 1995, Searle and Hedderson 2000, Kress et al. 2002, Ngamriabsakul et al. 2004, Takano and Nagamasu 2007). However, the distinc- tion, based on morphological characters, became ambiguous as several taxa described recently exhibit exceptions to the usual generic characters. For example, S. calcicola A.D.Poulsen & R.J.Searle, a species named in 2005 from Sarawak, has a distichous inflorescence, a distinguishing character for the genusDistichochlamys M.F.Newman. Larsen and Newman (2001) also reported another Scaphochlamys species with a dis- tichous inflorescence from north Peninsular Malaysia. A current study on the mor- phology of Scaphochlamys also revealed that some species display the characteristics of Distichochlamys and Myxochlamys. To test the validity of the current generic concept of Scaphochlamys and closely related genera Distichochlamys and Myxochlamys, we have examined their relationship by utilising ITS and matK markers together with the anal- ysis of the morphology across these genera. Borneocola (Zingiberaceae), a new genus from Borneo 33 Materials and methods Morphological study The morphological study was based on living plants in the forest, cultivated plants in the nursery of the Forest Research Institute Malaysia and specimens in the herbaria of AAU, BKF, C, FI, E, K, KEP, KLU, PSU, SAN, SAR and SING. A total of 372 herbarium specimens were examined in this study which includes 29 Scaphochlamys species and four Borneocola species (the types of another four Borneocola species were not yet deposited in the herbaria).The morphological characters examined in the study were habit; position of the rhizome, thickness and colour; height of leafy stem, its base (whether swollen to form a bulbous base); distance between leafy stems; characters of bladeless sheath such as colour, indumentum, number and length; ligule length, indumentum and shape; petiole length, indumentum, whether channelled or rounded in cross section; number of leaves per leafy stem; lamina colour on both surfaces, size, shape, venation, texture, indumentum, apex and base; length of the inflorescence and infructescence, arrangement of the floral bracts on the rachis, characters of floral bracts and bracteoles (colour, indumentum, texture, shape); size, colour and shape of calyx, floral tube, corolla lobes, labellum, staminodes, stamen, ovary. DNA extraction, amplification and sequencing Fresh leaves from the cultivated plants or silica-dried materials from plants collected in the field were used for genomic DNA extraction. For the ITS, the genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, California, USA) following the manufacturer’s protocol. Two primers, ITS 5P (5’-GGAAGGAGAAGTCGTAACAAGG-3’) and ITS 8P (5’-CACGCTTCTCCAGACTACA3’) (Moller and Cronk 1997) were used to am- plify the ITS region during the polymerase chain reaction (PCR). The thermal cycle of PCR for the amplification of the ITS sequences is initial denaturation at 94°C for 2 minutes, 40 cycles of denaturation at 94°C for 30 seconds, primers annealing at 48°C for 2 minutes, an extension at 72°C for 45 seconds and final extension at 72°C for 7 minutes. The PCR products were then purified using MinElute Gel Extraction Kit (Qiagen, Valencia, California, USA). For the matK, the protocols for DNA extraction, condition, purification and DNA sequencing were described previously by Takano and Nagamasu (2007). The PCR and sequencing primers for matK (cpDNA) were TA-240f (5’-GGGAAA GGATGGGGTCTCCCG-3’), TA-150r (5’-CTCAAGGAGTTTTGTGGTTC-3’), TA-470F (5’-CCCTCTCCCGTCCATATGGA-3’) (all three were designed in the present study), matK8 (Steele and Vilgalys 1994), m5r (Kress et al. 2002), matK8r (Ooi et al. 1995), trnK2621 (Liston and Kadereit 1995), TA-10F, TA-05R, TA-02F and TA-02R (all from Takano and Okada 2002). 34 Yen Yen Sam et al. / PhytoKeys 75: 31–55 (2016) Sequence alignment and phylogenetic analysis Raw sequence data were assembled and edited manually using BioEdit software ver. 7.2.5 (Hall 1999). DNA sequences were aligned with the CLUSTALW 1.83 software package, with default settings and multiple alignments (Thompson et al. 1994). Align- ments of the matK sequences of cpDNA and the ITS sequences of nrDNA were com- bined. Gaps were deleted. A total of 100 individuals including 54 taxa of Scaphochlamys and allied spe- cies were used. The three Siphonochilus species were used as an outgroup (Kress et al. 2002). Materials, accession numbers for the sequences, vouchers and references to the literature are presented in Table 1 at the end of this paper. Three datasets which comprise ITS, matK and ITS+matK combined, each containing 82, 78, and 61 taxa, were constructed. These three datasets were analysed using three methods: maximum parsimony, maximum likelihood and Bayesian analysis. A maximum parsimony (MP) analysis was performed with MEGA 6 (Tamura et al. 2013). Heuristic searches were conducted with RANDOM addition, SPR branch swap- ping and MULPARS options. Support for each branch was estimated with a boot- strap analysis, with 1000 replications (Felsenstein 1985), in a heuristic search